Abstract

Despite a growing understanding of the link between intestinal inflammation and resident gut microbes, longitudinal studies of human flora before initial onset of intestinal inflammation have not been reported. Here, we demonstrate in murine and human recipients of allogeneic bone marrow transplantation (BMT) that intestinal inflammation secondary to graft-versus-host disease (GVHD) is associated with major shifts in the composition of the intestinal microbiota. The microbiota, in turn, can modulate the severity of intestinal inflammation. In mouse models of GVHD, we observed loss of overall diversity and expansion of Lactobacillales and loss of Clostridiales. Eliminating Lactobacillales from the flora of mice before BMT aggravated GVHD, whereas reintroducing the predominant species of Lactobacillus mediated significant protection against GVHD. We then characterized gut flora of patients during onset of intestinal inflammation caused by GVHD and found patterns mirroring those in mice. We also identified increased microbial chaos early after allogeneic BMT as a potential risk factor for subsequent GVHD. Together, these data demonstrate regulation of flora by intestinal inflammation and suggest that flora manipulation may reduce intestinal inflammation and improve outcomes for allogeneic BMT recipients.

GVHD in mice produces marked changes in the microbiota. (A) B6 mice were lethally irradiated and transplanted with 5 × 106 B10.BR T cell–depleted BM supplemented with or without 1 × 106 splenic T cells. Features of GVHD are indicated on ileal histology sections from day 14, including lymphocytic infiltration (block arrows), crypt regeneration (enlarged crypts and hyperchromasia), and apoptosis (black arrows). Paneth cells are indicated (blue arrows). Bar, 20 µm. Representative images are shown from one of two independent experiments with similar results. Each dot represents an individual mouse, with bars indicating medians. (B) Quantitation of bacterial load of ileal contents on day 14 was performed by quantitative PCR of 16S rRNA gene copies. Results of a single experiment are shown. (C) Quantification of IgA levels in ileal contents on day 14 was performed by ELISA. Results of a single experiment are shown. (D) Comparison of representation by Unclassified Firmicutes, Barnesiella, and unclassified Porphyromonadaceae from ileal samples. Combined results from three experiments are shown. (E) Diversity of ileal floras from mice with GVHD was determined by the Shannon index. Combined results from two experiments are shown. (F) Principal coordinate analysis of unweighted UniFrac, of ileal floras from B6 mice transplanted with syngeneic (syn) or allogenic (allo) BM with or without T cells. Combined results from three experiments, with data points from each experiment indicated by number. Mice from experiment three were housed individually. (G) Dissimilarity of ileal floras of allo BMT recipient mice without and with GVHD compared with untreated mice by Bray-Curtis index. Combined results of two (Day 7) and 3 (Day 14) experiments are shown.

GVHD in mice produces marked changes in the microbiota. (A) B6 mice were transplanted with B10.BR donor BM and T cells as in Fig. 1. Comparison of representation by Lactobacillales, Clostridiales, and other Firmicutes from ileal samples. Combined results from three experiments are shown. (B) Comparison of representation by Lactobacillales, Clostridiales, and other Firmicutes from cecal samples. Combined results from two experiments are shown. (C) Bacterial composition at the genus level of ileal flora on day 14 after BMT are depicted with individual mice displayed in each bar. Results of three separate experiments, each displayed in a row, are shown. Additional untransplanted mice were treated with osmotic laxative or DSS starting on day 7 and also individually housed. (D) Mice were transplanted using the strain combinations indicated; mouse vendor was The Jackson Laboratory unless otherwise indicated. Bar graphs show bacterial composition of ileal contents at the genus level for individual mice on day 14.

Composition of intestinal flora can impact on severity of intestinal GVHD. (A) Schematic of treatment: B6 mice received ampicillin for 1 wk, followed by a 2-wk recovery period with unmodified drinking water; some were gavaged every 2 d with L. johnsonii (Lacto) of B6 flora origin during recovery, followed by harvest or BMT. Ileal contents were evaluated on days 0 (no BMT) and 14 after BMT. Bar graphs show bacterial composition of ileal contents at the genus level for individual mice. (B) Similar to as in A, BM12 mice received ampicillin followed by recovery; some also received L. johnsonii reintroduction. GVHD was induced upon transplantation with BM and either wild-type CD4 T cells (500K) or ABM RAG1 KO TCR transgenic CD4 T cells (100K). (C) B6 mice were treated with ampicillin, and then were or were not gavaged with L. johnsonii and transplanted with B10.BR BM and T cells. (top) Survival data combined from two experiments with similar results. (bottom) Pathological scores of GVHD target organs on day +21.

GVHD produces marked changes in the microbiota of humans, and the microbiota may affect risk of developing GVHD. (A) Summary of clinical parameters of non-GVHD and GVHD patients. (B) Flora diversity, by Shannon index, of stool samples after BMT. Individual measurements of diversity are displayed, as well as moving averages and P values calculated for 10-d intervals. (C) Contribution of bacterial populations in samples during two time periods, days 0 to 13 and 14 to 21 after BMT. (D) Microbial chaos of stool samples by mean Bray-Curtis time index from pre-BMT to day 13 after BMT.